The regulation of oxygen homeostasis is crucial to our existence as aerobes. At the cellular level, oxygen sensing is implicated in a large number of biomedically relevant processes, including the immune response, pre-natal lung development, erythropoiesis, and angiogenesis. When oxygen levels become too low (hypoxia), the hypoxia inducible factor (HIF) induces the expression of over 70 genes, controlling cellular O2 regulation. The key players in the [O2] response are two enzymes known as HIF-hydroxylases, which regulate the activity level of HIF. The long-range goal of this proposal is to understand the oxygen- dependent reactivity of the HIF-hydroxylases. The HIF-hydroxylases belong to the Fe(ll), ?-ketoglutarate dependent oxygenase superfamily. The two known varieties of HIF-hydroxylases are HIF-prolyl hydroxylase (PHD) and HIF-asparaginyl hydroxylase (FIH), which hydroxylate discrete domains of the ? subunit of HIF (HIF?). PHD and FIH are the primary O? sensors in humans, inactivating HIF? under conditions of normal or elevated [O2]. Consequently, understanding the mechanism of these enzymes may provide avenues to control cellular responses to O2 levels. While much is known about ?KG oxygenases in general, the molecular details of O2 activation remain largely speculative, and the structural link between substrate and O2 binding is unclear. We will apply biophysical, mechanistic, and spectroscopic methods to understand the oxygen-dependent activity of HIF hydroxylases and a mechanistically related enzyme, AtsK. Kinetic isotope effects, mutagenesis, and single- turnover kinetics will be used to delineate the O2 activation mechanism. We will combine mechanistic and solvent-accessibility studies to probe the chemical and structural relationship between substrate and O2 binding. Non-productive reactions with O2 will be explored, as they may represent a physiological inactivation pathway. This research will identify structures unique to the HIF hydroxylases which regulate their oxygen-dependent reactivity, as well as better understanding of O2-activation in the broad class of ?KG oxygenases.